The monitoring of various medical conditions often requires measuring the levels of various components within the blood. In order to avoid invasive repeated blood drawing, implantable sensors aimed at detecting various components of blood in the body have been developed. More specifically, in the field of endocrinology, in order to avoid repeated “finger-sticks” for drawing blood to assess the levels of glucose in the blood in patients with diabetes mellitus, implantable glucose sensors have been discussed.
One method for sensing the concentration of an analyte such as glucose relies on Fluorescence Resonance Energy Transfer (FRET). FRET involves the transfer of energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when the donor and acceptor are in close proximity to each other, leading to fluorescence emission by the acceptor. Because of the high sensitivity of the FRET signal to the relative proximity of the fluorophores it is often used in biological research as a measurement tool. For example, the concentration of an analyte such as glucose can be measured by creating a fused sensor which includes two fluorophores and a third moiety which has specific binding site for the analyte. The conformational change of the fused sensor which results from the binding of the analyte changes the distance between the fluorophores, affecting the FRET signal and thus enabling the measurement of the analyte concentration.
PCT Patent Application Publication WO 2006/006166 to Gross et al., which is incorporated herein by reference, describes a protein which includes a glucose binding site, cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP). The protein is configured such that binding of glucose to the glucose binding site causes a reduction in a distance between the CFP and the YFP. Apparatus is described for detecting a concentration of a substance in a subject, the apparatus comprising a housing adapted to be implanted in the subject. The housing comprises a fluorescence resonance energy transfer (FRET) measurement device and cells genetically engineered to produce, in situ, a FRET protein having a FRET complex comprising a fluorescent protein donor, a fluorescent protein acceptor, and a binding site for the substance.
An alternative approach to glucose sensing has been discussed e.g. by Y J Heo et al., in “Towards Smart Tattoos: Implantable Biosensors for Continuous Glucose Monitoring,” Adv. Healthcare Mater. 2013 January; 2(1):43-56 (Epub Nov. 26, 2012). Heo et al. provide a review of the efforts to develop analyte monitoring methods, which include placing a fluorescent material sensitive to a target analyte, e.g., glucose, under the skin and reading the optical signal through the skin, thus enabling measurement of the analyte.
In recent years, improved far-red fluorophores, having a significant portion of their emission spectrum above 650 nm, have been developed in order to exploit optical properties of biological tissue and enable in-vivo deep imaging, including, e.g., TagRFP, mRuby, mRuby2, mPlum, FusionRed, mNeptune, mNeptune2.5, mCardinal, Katushka, mKate, mKate2, mRaspberry and others. The relative emission of these fluorophores at an optical window above 650 nm is typically 10-50%, enabling sufficiently-effective detection through the skin. Additionally, infrared phytochromes such as iRFP, IFP1.4, and IFP2.0 have been developed which further push the emission spectrum into the infrared; however, these phytochromes depend on the availability of biliverdin, possibly complicating their practical use. Red fluorophores may effectively be used in conjunction with shorter-wavelengths fluorophores (e.g., green) to create FRET couples that can be used to develop different types of biosensors, as shown for example by Lam et al.